EP3849661A1 - Bispecific antibody targeting il-1r1 and nlpr3 - Google Patents

Bispecific antibody targeting il-1r1 and nlpr3

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Publication number
EP3849661A1
EP3849661A1 EP19769486.2A EP19769486A EP3849661A1 EP 3849661 A1 EP3849661 A1 EP 3849661A1 EP 19769486 A EP19769486 A EP 19769486A EP 3849661 A1 EP3849661 A1 EP 3849661A1
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EP
European Patent Office
Prior art keywords
antibody
nlrp3
seq
modulator
binding
Prior art date
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Pending
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EP19769486.2A
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German (de)
English (en)
French (fr)
Inventor
Victoria MCGILLIGAN
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Ulster University
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Ulster University
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Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to modulators of the NLRP3 inflammasome pathway, particularly antibodies and fragments thereof as well as aptamer molecules (small RNA/DNA molecules that can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets), each of which have binding specificity for members of the NLRP3 inflammasome.
  • the invention extends to use of such antibodies and aptamers, and their fragments, for the treatment and prevention of inflammatory diseases mediated by NLRP3 inflammasome signalling and activation, particularly inflammatory eye diseases such as glaucoma.
  • Inflammasomes are a group of protein complexes that recognize a large variety of inflammation inducing stimuli that include pathogen-associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs).
  • PAMPs pathogen-associated molecular patterns
  • DAMPs danger associated molecular patterns
  • NLRP3 is the most studied inflammasome due to the large variety of signals that activate it, including LPS, bacterial toxins, dust, stress signals such as ATP, crystallized and particulate materials, cholesterol crystals, oxidised LDL, amyloid beta, prion protein fibrils and fibrillar alpha synuclein, shear stress, pressure.
  • NLRP3 nucleotide-binding oligomerization domain (NACHT)
  • LRR leucine rich repeat
  • pyrin domain-containing protein 3 inflammasome is implicated in a number of infectious diseases and a plethora of degenerative inflammatory type diseases including Atherosclerosis, Diabetes, Inflammatory eye disease, other eye diseases such as dry eye syndrome, Glaucoma, Age related macular degeneration, Depression, Alzheimer’s Disease, Parkinson’s Disease, Inflammatory Bowel Diseases, Arthritic conditions such as Rheumatoid Arthritis, Ageing, Dermatological conditions and Cancer.
  • the main role of the NLRP3 protein is to sense danger signals or foreign material, and relay the signal to caspase 1 in turn activating the secretion of the pro-inflammatory cytokine I L- 1 b , which then initiates inflammation in an attempt to protect the body.
  • IL-1 b is the most studied of all cytokines because of its central role in the inflammatory process. Although it is useful for the body to activate I L- 1 b , in many diseases this inflammation can get out of control and be responsible for the pathogenesis of the disease. Most therapeutic strategies to date have concentrated on developing therapies against IL-1 b to dampen the inflammation, but as we propose here, there are number of advantages of targeting the upstream controllers of this cytokine, namely the NLRP3 inflammasome.
  • NFKB pathway is activated by a DAMP or PAMP via Toll-like receptors (TLRs) and or CD36 receptors. This leads to the transcription and expression of the pro form of IL-1 b and NLRP3.
  • TLRs Toll-like receptors
  • CD36 receptors CD36 receptors
  • a second signal is also thought to be required whereby purinergic receptor stimulation by a DAMP such as ATP leads to increases in intracellular calcium and cell swelling that results in potassium efflux from the cell, lysosomal destabilisation, membrane permeabillisation, mitochondrial damage and subsequent generation of reactive oxygen species, leading to NLRP3 activation.
  • DAMP purinergic receptor stimulation
  • ATP oxidized LDL cholesterol
  • oxidized LDL cholesterol can indeed itself act as the two signals required for NLRP3 activation.
  • potassium efflux appears to be the sole common denominator for NLRP3 activation.
  • the NLRP3 protein subsequently interacts with ASC (apoptosis-associated speck-like protein) through homotypic interactions of the pyrin domain.
  • ASC apoptosis-associated speck-like protein
  • ASC then interacts with pro caspase 1 resulting in cleavage and activation of caspase 1 , which in turn cleaves pro IL-1 b to its active form.
  • IL-1 b is then cleaved to produce the biologically active and secreted form.
  • NLRP3 is a nod like receptor so dampening the recognition of the root cause of a disease, i.e.
  • recognition of the foreign / danger material may be advantageous over dampening the response.
  • IL-1 b would be secreted via the NLRP3 pathway activated by disease specific stimuli, e.g. oxidized LDL, b amyloid or alpha synuclein or a particular pathogen.
  • disease specific stimuli e.g. oxidized LDL, b amyloid or alpha synuclein or a particular pathogen.
  • IL-1 b could still be activated via other pathways in response to other non-disease-related stimuli as needed in extreme circumstances (such as large scale or opportunistic infections), since there are other pathways responsible for IL-1 b activation.
  • the inflammasome has been associated with specialized forms of cell death, pyronecrosis
  • an anti-NLRP3 therapy will also decrease such death pathways, which have been evidenced to be involved in the pathogenesis of certain diseases such as atherosclerosis.
  • Pyroptosis is a risk factor for plaque disruption in this disease in response to oxidized LDL.
  • Glyburide a sulfonylurea drug
  • MCC950 is another example of a specific small molecule inhibitor of NLRP3 inflammasome:
  • NLRP3 also known as NALP3 and cryopyrin
  • NLRP3 is a cytosolic protein; therefore, in order to target this protein, any therapy must gain entry to the cell.
  • Humanized antibodies are quite large in size and entry to the cytosol may prove difficult.
  • Small antibody fragment development also present a possibility to overcome such a challenge where an antibody fragment may be a Fab fragment, which is the antigen-binding fragment of an antibody, or a single-chain variable fragment, which is a fusion protein of the variable region of heavy and the light chain of an antibody connected by a peptide linker.
  • the present inventor has devised additional strategies to ensure the therapeutic antibody or aptamer, and their fragments, can gain entry to the cell.
  • W02013/007763A1 discloses an inhibitor capable of intracellular localisation and cytosolic binding to a member of the inflammasome group including NLRP3, for use in a method for the prevention/treatment of acne.
  • US20080008652A1 discloses methods and compositions for modulating immune responses and adjuvant activity, and in particular, via modulation of cryopyrin (NPRL3) signalling.
  • NPRL3 cryopyrin
  • Humanized antibodies that target cryopyrin modulating proteins, or cryopyrin signal pathway components, are mentioned, and methods of producing cryopyrin antibodies are disclosed.
  • W02002026780A2 discloses antibodies that bind to PAAD-domain containing polypeptides, as well as methods of treating various pathologies, including inflammation, by administering an anti-PAAD antibody.
  • Single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof are also mentioned.
  • WO201 1 109459A2 discloses a method of treating an inflammatory disease of skin/hair by providing a composition including at least one antibody that specifically binds to a component(s) of a mammalian inflammasome, such as ASC or NLRP1 .
  • a mammalian inflammasome such as ASC or NLRP1 .
  • Commercially available antibodies to ASC and NPRL1 are mentioned.
  • EP2350315B1 discloses methods and kits for the early diagnosis of atherosclerosis, involving the measurement of the expression levels of NLRP3, ASC and/or caspase-1. Expression levels may be measured by methods involving antibodies, including human antibodies, humanized antibodies, recombinant antibodies and antibody fragments, which in turn include Fab, Fab', F(ab)2, F(ab')2, Fv and scFv.
  • WO20131 19673A1 discloses a method of evaluating a patient suspected of having a CNS injury comprising measuring the level of at least one inflammasome protein such as NLRP1 (NALP-1 ), ASC, and caspase-1.
  • NLRP1 NLRP1
  • ASC ASC
  • caspase-1 NLRP1
  • Commercially available antibodies to NPRL-1 , ASC and caspase-1 are mentioned.
  • W02007077042A1 discloses a method for the treatment of gout or pseudogout, comprising administering a NALP3 inflammasome inhibiting agent.
  • the NALP3 inflammasome inhibiting agents are described as acting downstream of the NALP3 inflammasome and selected from among antibodies that inhibit the activity of IL-1.
  • WO2013138795A1 discloses a fusion protein comprising a Surf+ Penetrating Polypeptide and an antibody or antibody-mimic moiety (AAM moiety) that binds to an intracellular target, wherein the fusion protein penetrates cells and binds to the intracellular target to inhibit binding between the target and another protein inside the cells.
  • AAM moiety an antibody or antibody-mimic moiety
  • the present invention provides novel and effective modulators of the NLRP3 inflammasome for the treatment and prevention of inflammatory diseases mediated by NLRP3 inflammasome signalling and activation, particularly inflammatory eye diseases such as glaucoma.
  • modulators include a bi-antibody or aptamer, and their fragments, targeted to both of IL-1 R1 and NLRP3.
  • the bi-antibody first gains entry into the cell by binding to the IL-1 R1 which triggers rapid internalisation and, once internalised, the bi-antibody then targets the intracellular protein NLRP3 inhibiting the assembly of the NLRP3 inflammasome, in turn preventing IL-1 b secretion from the cells, and reducing the initiation/amplification of inflammation.
  • an NLRP3 inflammasome modulator which is capable of binding to both of IL-1 R1 and NLRP3 for use in the treatment or prophylaxis of an inflammatory eye disease.
  • the inflammatory eye disease is glaucoma.
  • the modulator is also capable of binding to the PYD domain of NLRP3.
  • the modulator is selected from the group comprising: a polyclonal antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody, a fusion protein, or an aptamer molecule, a combination thereof, and fragments of each thereof.
  • the modulator may be a bi-antibody capable of binding to both of : IL-1 R1 and NLRP3.
  • the modulator is a recombinant humanized bi-antibody capable of binding to both of: IL-1 R1 and NLRP3.
  • the modulator is a bi-antibody comprising one or more of the binding regions of a first antibody capable of binding IL-1 R1 and one or more of the binding regions of a second antibody capable of binding NLRP3.
  • the modulator is a bi-antibody comprising one or more complementary determining regions (CDRs) of a first antibody capable of binding IL-1 R1 and one or more CDRs of a second antibody capable of binding NLRP3.
  • the first and/or second antibody is a monoclonal antibody.
  • the modulator is selected from an antibody fragment capable of binding to both: IL-1 R1 and NLRP3.
  • the antibody fragment is selected from one or more of Fab, Fv, Fab’,
  • the modulator is selected from a recombinant humanized antibody or antibody fragment capable of binding to both of: IL-1 R1 and NLRP3.
  • the modulator is an antibody or antibody fragment raised against one or more antigens selected from both of IL-1 R1 and NLRP3.
  • the modulator is raised against one or more antigens selected from all or part of both of IL-1 R1 and NLRP3.
  • the modulator is raised against one or more antigens selected from NLRP3, optionally conjugated to a carrier protein such as Keyhole Limpet Haemocyanin (KLH) (hereinafter, the NLRP3 immunogen), and IL-1 R1 , optionally recombinant IL-1 R1.
  • KLH Keyhole Limpet Haemocyanin
  • the extracellular domain of IL-1 R1 (hereinafter, the IL-1 R1 immunogen) comprises the sequence:
  • SRSPGK* (SEQ ID NO: 1 ).
  • the NLRP3 immunogen comprises the sequence:
  • the NLRP3 immunogen comprises a carrier protein conjugated to the sequence
  • EDYPPQKGCIPLPRGQTEKADHVD (SEQ ID NO: 30), optionally conjugated to the N-terminal end of the sequence EDYPPQKGCIPLPRGQTEKADHVD (SEQ ID NO: 30).
  • a carrier protein, conjugated to a peptide, is known in the art to help the peptide generate a stronger immune response.
  • the carrier protein is KLH.
  • the carrier protein is conjugated to the sequence EDYPPQKGCIPLPRGQTEKADHVD (SEQ ID NO: 30) via a linker, optionally the linker is Hydrazide-Ahx.
  • the NLRP3 immunogen is:
  • a hydrazide is a class of organic compounds characterized by a nitrogen- nitrogen covalent bond with four substituents with at least one of them being an acyl group.
  • Ahx denotes a 6-carbon linear aminohexanoic linker.
  • the modulator is raisable, optionally raised, against one or more immunogens selected from NLRP3 immunogen and IL-1 R1 immunogen, wherein the IL-1 R1 immunogen comprises the sequence:
  • SRSPGK* (SEQ ID NO: 1 ).
  • NLRP3 immunogen comprises the sequence:
  • the modulator is a bi-antibody comprising one or more of the binding regions of a first antibody raisable, optionally raised, against IL-1 R1 immunogen and comprising the sequence:
  • the modulator is a bi-antibody comprising one or more complementary determining regions (CDRs) of a first antibody raisable, optionally raised, against IL-1 R1 immunogen and comprising the sequence:
  • the first and/or second antibody is a monoclonal antibody.
  • the consensus sequence of the heavy chain of the first antibody (to IL-1 R1 ) is
  • the heavy chain CDRs of the first antibody comprise: GYPFTTAG (SEQ ID NO: 60); MNTQSEVP (SEQ ID NO: 61 ); and AKSVYFNWRYFDV (SEQ ID NO: 62).
  • the consensus sequence of the light chain of the first antibody (to IL-1 R1 ) is
  • the light chain CDRs of the first antibody comprise: QSISDY (SEQ ID NO: 63); YAS; and QHGHSFPLT (SEQ ID NO: 64).
  • the consensus sequence of the heavy chain of the second antibody (against NLRP3) is
  • the heavy chain CDRs of the second antibody comprise: GFTFSDYY (SEQ ID NO: 65); ISDGGTYT (SEQ ID NO: 66); and ARGWVSTMVKLLSSFPY (SEQ ID NO: 67).
  • the consensus sequence of the light chain of the second antibody (to NLRP3) is
  • SPSVTLFPPSTEELSL (SEQ ID NO: 43).
  • the light chain CDRs of the second antibody comprise: TGAVTTSNY (SEQ ID NO: 68); GTN; and ALWYSNYWV (SEQ ID NO: 69).
  • the modulator is capable of binding simultaneously to IL-1 R1 and NLRP3.
  • the modulator is capable of binding sequentially to IL-1 R1 and NLRP3.
  • the light chain of a bi-specific antibody of the present invention has the amino acid sequence:
  • the heavy chain of a bi-specific antibody of the present invention has the amino acid sequence:
  • PK** (SEQ ID NO: 59). By“binding simultaneously” to both of IL-1 R1 and NLRP3, it is meant that the modulator is capable of binding to each of IL-1 R1 and / or NLRP3, whether said IL-1 R1 and / or NLRP3 are formed as a complex, or whether they are not formed as a complex.
  • the invention provides an NLRP3 inflammasome modulator as defined herein in relation to the first aspect of the invention for use in the treatment or prophylaxis of an inflammation- related disorder, optionally an inflammatory eye disease, such as glaucoma, as described in the first aspect of the invention, in which the NLRP3 inflammasome is known to play a key role in the disease pathogenesis.
  • bispecific antibody as the modulator is that it can be used at lower, and thus less toxic, concentrations than single antibodies, therefore, reducing cytotoxicity potential. Being bispecific allows for a more stable antibody with greater purity.
  • the present invention provides a method for the treatment and/or prophylaxis of an inflammation-related disorder, optionally an inflammatory eye disease, such as glaucoma, the method comprising the steps of: providing a therapeutically effective amount of an NLRP3 inflammasome modulator as defined herein in relation to the first aspect of the invention which suppresses activation and/or signalling of the NLRP3 inflammasome, and
  • the present invention provides for use of an NLRP3 inflammasome modulator as defined herein in relation to the first aspect of the invention in the preparation of a medicament for the treatment of an inflammation-related disorder, optionally an inflammatory eye disease, such as glaucoma.
  • the present invention provides a method to reduce or prevent or treat at least one symptom of an inflammation-related disorder, optionally an inflammatory eye disease, such as glaucoma, in a subject comprising selectively inhibiting and / or reducing activation of the inflammasome pathway by the use of an NLRP3 inflammasome modulator as defined herein in relation to the first aspect of the invention.
  • the modulator is for use in the treatment or prevention of at least one symptom of an inflammation-related disorder in a subject comprising selectively inhibiting and or reducing activation of the inflammasome pathway by the use of the modulator.
  • the light chain of a bi-specific antibody has the amino acid sequence of SEQ ID NO: 57 and the heavy chain of a bi-specific antibody the amino acid sequence of SEQ ID NO: 59 and may be referred to herein as InflaMab or Inflamab.
  • InflaMab may have disease modifying effects in systemic conditions such as but not limited to Atherosclerosis, whereby it prevents/inhibits inflammation therefore preventing plaque build up and/or plaque rupture thus reducing risk of myocardial infarction.
  • InflaMab may have disease modifying effects in eye diseases such as but not limited to Glaucoma, whereby it prevents/inhibits inflammation, reduces intraocular pressure and / or prevents loss of retinal ganglion cells and axons, protecting the optic nerve and preserving visual acuity, and/or preventing blindness.
  • eye diseases such as but not limited to Glaucoma, whereby it prevents/inhibits inflammation, reduces intraocular pressure and / or prevents loss of retinal ganglion cells and axons, protecting the optic nerve and preserving visual acuity, and/or preventing blindness.
  • InflaMab may have disease modifying effects in neurological conditions such as but not limited to Alzheimer’s Disease, whereby it prevents/inhibits inflammation, reduces/inhibits amyloid plaque load, and/or prevents of cognitive dysfunction.
  • the modulator as defined herein may have utility in individuals with multi-morbidities or comorbidities associated with inflammation.
  • the modulator as defined in relation to of any of the aforementioned aspects of the invention is a 210 kiloDalton (kDa) bispecific mouse antibody composed of two pairs of light chain and two pairs of heavy chains with scFv domains fused to the N-terminal, complexed together via disulphide bonds.
  • an“inflammation-related disorder” includes, but is not limited to, Atherosclerosis, inflammatory eye conditions such as Age-Related Macular degeneration, Dry Eye Syndrome, Glaucoma, Sjogren’s syndrome, Diabetes, Inflammatory eye disease, Depression, Alzheimer’s Disease, Parkinson’s Disease, Inflammatory Bowel Disease, Rheumatoid Arthritis, Ageing,
  • the subject is a mammal, such as a human.
  • antibody should be construed as covering any binding member or substance having a binding domain with the required specificity.
  • the antibody of the invention may be a monoclonal antibody, or a fragment, functional equivalent or homologue thereof.
  • the term includes any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Fragments of a whole antibody can perform the function of antigen binding.
  • binding fragments are; a Fab fragment comprising of the VL, VH, CL and CH1 antibody domains; an Fv fragment consisting of the VL and VH domains of a single antibody; a F(ab’)2 fragments, a bivalent fragment comprising two linked Fab fragments; a single chain Fv molecule (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; or a bi- or tri-specific antibody, which may be multivalent or multispecific fragments constructed by gene fusion.
  • a fragment of an antibody or of a polypeptide for use in the present invention generally means a stretch of amino acid residues of at least 5 to 7 contiguous amino acids, often at least about 7 to 9 contiguous amino acids, typically at least about 9 to 13 contiguous amino acids, more preferably at least about 20 to 30 or more contiguous amino acids and most preferably at least about 30 to 40 or more consecutive amino acids.
  • antibody includes antibodies which have been "humanized”. Methods for making humanised antibodies are known in the art.
  • Aptamers are peptide molecules that bind to specific target molecules. Aptamers are in the realm between a small molecule and a biologic. They exhibit significant advantages relative to antibody therapeutics in terms of size, synthetic accessibility and modification.
  • Modulators as described herein may be used in assays, such as ELISAs, to detect NLRP3 from human blood or tissue samples.
  • the present invention provides a kit comprising one or more modulators of the first aspect of the invention.
  • the kit further comprises instructions for use of said kit.
  • the kit is for detecting NLRP3 in human cells, in blood or tissue samples.
  • Figure 1 4-20% denaturing, reducing and non-reducing, SDS-PAGE analysis of IL-1 R1 FC.
  • Figure 7 24 Well-Plate Screening Results.
  • Figure 8 Final Selected Flybridomas from F237 5D1-1A8.
  • Figure 9 Final Selected Flybridomas from F237 5D1-1A8 final 24w Screening.
  • Figure 10 IL-1 R1 Internalisation in THP1 cells- immunofluorescence imaging. Fluorescence microscopic images taken from THP1 macrophages treated with LPS and ATP to induce the expression of the IL-1 R1.
  • FIG. 11 IL-1 R1 Internalisation in THP1 cells- flow cytometry.
  • Figure 12 PCR using several combinations of Ig variable domain primers.
  • Figure 13 Graphical representation of the CDR loops. Ref: Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501 ).
  • Figure 14 Graphical representation of the CDR loops (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501 ).
  • Figure 15 Structure of NLRP3 inflammasome. Bergsbaken, T.; Fink, S. L.; Cookson, B. T. (2009). "Pyroptosis: Host cell death and inflammation”. Nature Reviews Microbiology. 7 (2): 99-109.
  • Figure 16 Sequence alignment using CLUSTAL 0 (1.2.4) of the consensus sequences of C-term domains of human and mouse NALP (NLRP) proteins.
  • Figure 17 Novafold predicted structure of Peptide FUS_746_001 (Yellow) aligned to NLRP3 PDB: 3QF2 showing secondary structural features using Protean 3D, version 14.0.1
  • Figure 18 Immunized mice expressed high levels of the NLRP3 mAb.
  • Figure 21 Post Fusion Screening Results.
  • Figure 24 24 Well-Plate Screening Results.
  • FIG. 25 Final Selected Flybridomas from F226.
  • FIG. 27 Western Blot Analysis.
  • Figure 28 PCR using several combinations of Ig variable domain primers.
  • Figure 29 Graphical representation of the CDR loops (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501 ).
  • Figure 30 Graphical representation of the CDR loops (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) PMID: 12477501 ).
  • Figure 31 Diagram illustrating the bispecific design and the plasmid map of InflaMab.
  • Figure 32 4-20% SDS-PAGE analysis of InflaMab. Molecular weight marker shown in kiloDaltons.
  • Figure 33 Inflamab prevents IL-1 b release. (Note,“Ulster Ab” is synonymous with“Inflamab” and “Bi-specific Ab”.)
  • Figure 34 Inflamab prevents caspase-1 activation in THP1 cells.
  • Figure 35 Internalization of Inflamab.
  • Figure 36 Constitutive expression of NLRP3 in the mouse and human ONH.
  • Figure 37 Constitutive NLRP3 expression in the astrocytes of the human ONH.
  • Figure 38 Assembly of the NLRP3 inflammasome in the ONH coincides with the induction of inflammatory mediators at 7 days post microbead injection.
  • FIG 39 Early induction of inflammatory mediators and accumulation of Iba1+ cells in the ONH is abrogated in inflammasome deficient (ASC KO) mice.
  • Figure 40 ASC and NLRP3 are required for lOP-induced axon degeneration and death of RGCs in microbead-induced mouse model of glaucoma.
  • Figure 41 NLRP3 small molecule inhibitor, MCC950, prevents death of RGCs in microbead model of glaucoma.
  • Figure 42 InflaMab prevents death of RGCs in microbead model of glaucoma.
  • the modulator of the invention e.g. the bi-specific antibody, acts according to steps which include:
  • Targeting the antibody to IL-1 R1 triggers internalisation of the IL-1 R1 , thus making less IL- 1 R1 available for IL-1 b binding resulting in further inhibiting the potentiation and amplification of inflammation.
  • Such a modulator of the first aspect of the invention provides a surprisingly additive inhibitory effect upon the inflammasome as a whole, not only the NLRP3 protein portion and thus will provide a more effective inhibitor of inflammasome-related diseases.
  • Example 1 Transient Expression of IL-1R1 Fc fusion
  • IL-1 R1 Fc is transiently expressed and purified in HEK293 cells.
  • the purified protein is evaluated for size and purity by SDS PAGE and tested for endotoxin levels. Finally the protein is evaluated for activity by ELISA.
  • a mammalian expression vector encoding interleukin-1 receptor (IL-1 R1 ) Fc fusion protein was transfected into HEK293 cells.
  • the expressed Fc fusion protein was subsequently purified from cell culture supernatant using standard chromatography techniques. The concentration and purity were determined for the purified product.
  • DNA coding for the amino acid sequence of IL-1 R1 Fc was synthesised and cloned into a mammalian transient expression plasmid pD2610-v1 (DNA2.0).
  • IL-1 R1 Fc was expressed using a HEK293 cell based transient expression system and the resulting antibody containing cell culture supernatants was clarified by centrifugation and filtration.
  • Two lots of IL-1 R1 Fc were purified (using AKTA chromatography equipment) from cell culture supernatants via protein A affinity chromatography. Purified protein was dialysed/buffer exchanged into phosphate buffered saline solution.
  • Figure 1 shows 4-20% denaturing, reducing and non-reducing, SDS-PAGE analysis of IL-1 R1 FC. Molecular weight marker shown in kiloDaltons. Lanes are as follows:
  • Example 1A IL-1R1 Fc amino acid sequence MKVLLRLICFIALLISSLEADKCKEREEKIILVSSANEIDVRPCPLNPNEHKGTITWYKDDSKTPVSTEQ
  • the aim of this project is to generate a monoclonal antibody against IL-1 R1.
  • a population of 5 mice were immunised and screened for positive immune responses. After selecting a suitable candidate for fusion, splenocytes were fused with partner cells to produce a population of hybridomas. This population underwent a series of limiting dilutions and screening assays to produce fully monoclonal cell lines.
  • the product name“F237 5D1-1A8-2A5” refers to one of the 10 chosen monoclonal hybridoma cell lines.
  • the name is comprised of components describing the production pathway at each stage.
  • Each hybridoma selected from the post-fusion screening and each limiting dilution was given a number corresponding to the plate number and well location on that plate for which the hybridoma was chosen (i.e. 5D1-1A8-2A5). This nomenclature traces the derivation of each individual hybridoma allowing for clear differentiation in the screening process.
  • mice A population of 5 BalbC mice were immunised subcutaneously with 200 pi of a 1 :1 emulsion of Freund’s Adjuvant Complete (Sigma) and a 600 pi aliquot of IL-1 R1 prepared herein.
  • the population was immunized with a 2 nd injection at the same volumes and concentrations as the original injection only using Freund’s Adjuvant Incomplete (Sigma) instead.
  • the mice were tagged by ear punches (NP, RP, LP, LRP, 2LP), and test bleeds were screened as described herein for preliminary results.
  • Tail bleeds were taken from the population of 5 BalbC mice and centrifuged at 8000 rpm for 10 min at RT (room temperature). The blood serum from each mouse was collected, loaded onto the plate the same day as screening, and stored at -20°C. This screening was performed twice for the selection of a suitable mouse for fusion.
  • a Maxi Sorp plate was coated by adding 100 mI/well of 50 mM sodium carbonate coating buffer (pH 9.5) containing the IL-1 R1 at 1 pg/ml.
  • a separate coating solution was prepared by diluting APO-A1 in the same coating buffer at 1 pg/ml. These solutions were loaded onto the plate in alternating rows so as to provide two wells to load each sample that demonstrates a positive and negative result. This plate was incubated overnight at 4°C in static conditions.
  • the mouse with an ear tag of RP was selected for fusion by expressing the most positive immune response.
  • SP2 cells were broken out from liquid nitrogen and were passaged in 10% FCS DMEM supplemented with 1 % Pen/Strep, 1 % L-glutamine until 3x12 ml T75 flasks were 75%- 90% confluent on the day of fusion.
  • FCS DMEM 10% FCS DMEM supplemented with 1 % Pen/Strep, 1 % L-glutamine until 3x12 ml T75 flasks were 75%- 90% confluent on the day of fusion.
  • SP2 cells were dislodged by tapping the flask and were centrifuged at 1000 rpm for 5 min at 37°C. The cells were resuspended in 20 ml SFM DMEM, centrifuged again, and resuspended in 10 ml SFM DMEM.
  • SP2 cells were stored in a Sterilin tube in SFM at 37°C, 6% C0 2 until needed.
  • the spleen was aseptically removed from the mouse that showed the strongest immune response.
  • Splenocytes were extracted by puncturing both ends of the spleen with a fine gauge needle and flushing 10-15 ml SFM DMEM.
  • Splenocytes were transferred to a sterilin tube and washed twice with 20 ml serum free DMEM by centrifugation at 1300 rpm for 5 min at 37°C and gently removing the supernatant.
  • the splenocytes were resuspended in 10 ml Serum free DMEM in a sterilin tube.
  • the SP2 cells were added to the splenocytes. This
  • SP2/splenocytes culture was centrifuged at 1300 rpm for 5 min at 37°C. After discarding the supernatant, 1 ml PEG was added to the SP2/splenocytes culture dropwise while stirring continuously over a period of 3 min. 1 ml SFM DMEM was added to the fusion mixture and stirred for 4 min. 10 ml SFM DMEM was added dropwise to the fresh culture and incubated for in 37°C water bath for 5 min. The cells were then centrifuged at 1000 rpm for 5 min at 37°C. The pellet was resuspended in 200 ml_ HATR media and was plated at 200 mI/well in 10x 96 well culture plates which were incubated 1 1 days at 37°C in 6% C0 2 prior to screening.
  • the samples from each dilution plate were loaded onto the ELISA plates at 100 mI/well by adding 1 row from each dilution plate per 2 rows on the ELISA plates to account for specificity of the coating antigens.
  • Two wells per ELISA were incubated with 100 mI 1xPBS as a negative control. These samples were incubated at 150 rpm for 2 hours at room temperature.
  • a secondary screen was performed to select the most specific and highest producing populations for rounds of limiting dilutions. Both limiting dilutions were performed for 1-3 protoclones each by seeding 2-4x 96-well plates at 1 cell/ well in 200 pi culture/well. The plates were prepared by counting each culture in the 24-well plate and were diluted 10x as an intermediate dilution, then were diluted to 200 cells in 40 ml. The culture was plated at 200 mI/well and left to incubate at 37°C, 6% C0 2 for 7-10 days until the wells were 80%-90% confluent. Each well for both limiting dilutions were screened by ELISA as described herein.
  • each clone was left to grow in 37°C, 6% C0 2 for 6 days until each well became 80%-90% confluent.
  • each clone at 1 ml/well was transferred to a T25 flask containing 5 ml fresh 10% HATR DMEM for cryopreservation.
  • each 5 ml culture was centrifuged at 1000 rpm for 5 min at 37°C and was resuspended in 1 ml of fresh 10% DMEM HATR media.
  • Each 1 ml culture was transferred to a cryovial containing 300 mI of a 1 : 1 ratio of FCS to DMSO. The vials were sealed and placed in a Mr. Frosty and transferred to the -70°C freezer for short-term storage.
  • Anti-IL-1 R1 produced from clone F237 5D1-1A8-2A5 was selected for sequencing. Once the culture was confluent in the T25 flask, the supernatant was discarded. The cells were dislodged by cell scraping into 2 ml fresh media and were centrifuged at 7,600 rpm for 5 min at RT. The supernatant was then discarded and the pellet was flash frozen in liquid nitrogen and placed in -70°C until ready for mRNA extraction.
  • mice were immunised with an IL-1 R1 immunogen (produced in house in CHO cells) and regular test bleeds were taken over an 1 1 week period.
  • the mouse with an ear tag of RP was selected for the fusion of its splenocytes to fusion partner SP2 culture as it demonstrated the best immune response - see Figure 3.
  • Clones were selected from the post-fusion screening and were arrayed into a 24 well plate for expansion followed by a secondary screening that determines suitable protoclones for the first round of limiting dilutions - see Figure 5.
  • the limiting dilution was screened by ELISA against IL-1 R1 and APO-A1. Eleven hybridoma populations were selected from F237 2H12, F237 5D1 , and F237 7E6 that demonstrated the highest and most specific response - see Figure 6.
  • each clone was screened by ELISA against IL-1 R1 and APO-A1.
  • F237-5D1-1A8 was selected for the 2 nd round of limiting dilution over 4x 96 well plates - see Figure 7.
  • the cells were incubated with mouse serum from several different mice, containing the test antibody against the IL-1 R1 , which was conjugated to a pHrodoTM dye (that will only fluoresce within a cell). Strong IL-1 R1 immunoreactivity was observed in the nucleus and cytoplasm of the THP1 cells. IL-1 R1 and DAPI staining at X40 magnification. No staining was observed in the secondary antibody only treated control cells. Images are from four different wells used in two different experiments. The best mouse was selected to take forward to the fusion hybridoma and cloning stages.
  • THP1 macrophages (see Figure 11 ) treated with LPS and ATP to induce the expression of the IL- 1 R1.
  • the cells were incubated with mouse serum from several mice containing the test monoclonal antibody against the IL-1 R1 , which was conjugated to a pHrodo dye (that will only fluoresce within a cell) and analysed with flow cytometry. More fluorescence was seen in the IL-1 R1 antibody treated cells (i) as compared to the control secondary antibody only treated cells (ii). Using this data and that from Figure 3, the best mouse was chosen to take forward to the fusion hybridoma and cloning stages.
  • the aim of the project was to produce a range of antibodies against IL-1 R1.
  • RP was selected for fusion.
  • 10 monoclonal hybridoma cell lines were produced from two rounds of limiting dilutions. Each population was selected by highest production and highest specificity for IL-1 R1. These final cell lines have been frozen down, and the antibody expressed by this cell line will be sequenced.
  • Example 3 IL-1R1 monoclonal antibody sequencing mRNA was extracted from the hybridoma cell pellets. Total RNA was extracted from the pellets using a conventional RNA extraction protocol. Cell pellets were homogenised using RNA STAT-60 reagent. Upon addition of chloroform, the homogenate separated into an aqueous phase and an organic phase, and total RNA was isolated in the aqueous phase. Isopropanol was used to precipitate the RNA, followed by ethanol washes and solubilisation in water.
  • cDNA was created from the RNA by reverse-transcription with an oligo(dT) primer. PCR reactions are set up using variable domain primers to amplify both the VH and VL regions of the monoclonal antibody DNA giving the following bands - see Figure 12.
  • VH and VL products were cloned into the Invitrogen sequencing vector pCR2.1 and transformed into TOP10 cells and screened by PCR for positive transformants. Selected colonies were picked and analyzed by DNA sequencing on an ABI3130xl Genetic Analyzer, the result may be seen below.
  • VHl . 1 (51) AGLQWVQKMSGKGLKWIGWMNTQSEVPKYAEEFKGRIAFSLETAASTAYL VHl . 4 (51) AGLQWVQKMSGKGLKWIGWMNTQSEVPKYAEEFKGRIAFSLETAASTAYL VHl . 3 (51) AGLQWVQKMSGKGLKWIG MNTQSEVPRYAEEFKGRIAFSLETAASTAYL VH2. 1 (51) AGLQ VQKMSGKGLKWIGWMNTQSEVPRYAEEFKGRIAFSLETAASTAYL VH2. 5 (51) AGLQWVQKMSGKGLKWIGWMNTQSEVPKYAFEFKGRIAFSLETAASTAYL VH2.
  • VH1.1 (SEQ ID NO: 2)
  • VH1.4 (SEQ ID NO: 3)
  • VH1.3 (SEQ ID NO: 4)
  • VH2.1 (SEQ ID NO: 5)
  • VH2.5 (SEQ ID NO: 6)
  • VH2.3 (SEQ ID NO: 7)
  • VH1.2 (SEQ ID NO: 8)
  • VH2.4 (SEQ ID NO: 9)
  • GAGTTVTVSSAKTTPPPVYPLA SEQ ID NO: 7
  • variable domain is highlighted in BOLD.
  • CDRs Complementarity Determining Regions
  • Blue shaded circles are hydrophobic (non-polar) residues in frameworks 1-3 at sites that are hydrophobic in the majority of antibodies.
  • Red amino acids in the framework are structurally conserved amino acids.
  • VKl . 1 (101) EDVGVYYCQHGHSFPLIFGSGIKLELKRADAAPTVSIFPPSSEQLTSGGA VKl . 5 (101) EDVGVYYCQHGHSFPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGA VKl . 3 (101) EDVGVYYCQHGHSFPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGA VKl . 4 (101) EDVGVYYCQHGHSFPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGA VK2. 1 (101) EDVGVYYCQHGHSFPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGA VK2. 6 (101) EDVGVYYCQHGHSFPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGA Consensus (101) EDVGVYYCQHGHSFPLTFGSGTKLELKRADAAPTVSIFPPSSEQLTSGGA
  • VK1.1 (SEQ ID NO: 10)
  • VK1.5 (SEQ ID NO: 1 1 )
  • VK1.3 SEQ ID NO: 12
  • VK1.4 (SEQ ID NO: 13)
  • VK2.1 (SEQ ID NO: 14)
  • VK2.6 (SEQ ID NO: 15)
  • variable domain is highlighted in BOLD.
  • CDRs Complementarity Determining Regions
  • Blue shaded circles are hydrophobic (non-polar) residues in frameworks 1-3 at sites that are hydrophobic in the majority of antibodies.
  • Red amino acids in the framework are structurally conserved amino acids.
  • the NLRP-3 inflammasome is a heterogenous protein complex that forms in mammalian cells in response to inflammatory stimulus, the ability to regulate and attenuate its formation could have important therapeutic potential for a range of inflammatory disorders.
  • a peptide will be designed, derived from the NALP3 protein sequence which should generate antibodies capable of blocking the binding of NALP3 to the other protein components in the NLRP3 inflammasome complex.
  • NLRP3 activation occurs by the self-assembly of NLRP protein with ASC, which is a hetero-complex of CARD, PYD and Caspase-1 domains.
  • NLRP3 and ASC interact through their respective PYD domains, which contain a large proportion of highly conserved charged amino acid residues which interact to form electrostatic interactions, which stabilize the complex - see Figure 15.
  • Figure 16 shows sequence alignment using CLUSTAL 0 (1.2.4) of the consensus sequences of C- terminal domains of human and mouse NALP (NLRP) proteins. Red indicates a residue recognized as essential for NLRP/ASC interaction by site-directed mutagenesis (Vajjhala et al, 2012).
  • Peptide selection was concentrated on the sequence region from 1-61 which has been studied extensively and is involved in the interaction with ASC (Vajjhala et al, 2012). The region has also been well modelled by crystallography, with a number of PDB structures available for this domain.
  • PDB model 3QF2 which consists of the PYR domain of NLRP3 was selected as the most useful PDB structural reference.
  • Initial peptide candidate sequences were selected on the basis of accessibility and visibility as potential epitopes, and also degree of similarity between mouse and human sequence, whilst maintaining difference with other NLRP variants. These initial 3 peptides were modelled into 3D structures using NovaFold.
  • NovaFold is a 3D protein modelling software that uses the l-TASSER algorithm, a combination of template based threading (from PDB) and ab initio methods to predict the folding of a protein or peptide. It is used in this context to predict the presence of secondary structural features within a peptide which are known to be exhibited by the sequence in situ within the parent protein. This can help optimise the selection of a peptide sequence which best reflects the folding and proximity based relationships within the parent protein, helping to maximize the potential of the immunogenic protein resulting in an antibody with full activity towards the corresponding epitope in the full length protein.
  • Table 2 Comparison of alignment and structural features of peptide candidates modelled using Novafold 12.0 and aligned to NLRP3 structure PDB: 3QF2 using Protean 3D, version 14.0.1.
  • peptide FUS_746_001 is the preferred candidate for use as a peptide immunogen.
  • it also demonstrates high similarity in prediction of secondary structure and is an accessible epitopic target.
  • the NLRP3 peptide was synthesised by bioSynthesis Inc, Texas, conjugated to KLH using maleimide coupling through an additional C-terminal cysteine residue.
  • mice were immunised and screened for positive immune responses. After selecting a suitable candidate for fusion, splenocytes were fused with partner cells to produce a population of hybridomas. This population underwent a series of limiting dilutions and screening assays to produce fully monoclonal cell lines.
  • the product name“F226 7A7-1 E1-2D5” refers to one of the 10 chosen monoclonal hybridoma cell lines.
  • the name is comprised of components describing the production pathway at each stage.
  • Each hybridoma selected from the post-fusion screening and each limiting dilution was given a number corresponding to the plate number and well location on that plate for which the hybridoma was chosen (i.e. 7A7-1 E1-2D5). This nomenclature traces the derivation of each individual hybridoma allowing for clear differentiation in the screening process.
  • mice A population of 5 BalbC mice were immunised subcutaneously with 200 mI of a 1 :1 emulsion of Freund’s Adjuvant Complete (Sigma) and a 600 mI aliquot of NLRP3 peptide-KLH conjugate prepared herein.
  • Adjuvant Incomplete a 6 nd injection at the same volumes and concentrations as the original injection only using Freund’s Adjuvant Incomplete (Sigma) instead.
  • the mice were tagged by ear punches (NP, RP, LP, LRP, 2LP), and test bleeds were screened as described herein for preliminary results.
  • Tail bleeds were taken from the population of 5 BalbC mice and centrifuged at 8000 rpm for 10 min at RT. The blood serum from each mouse was collected, loaded onto the plate the same day as screening, and stored at -20°C. This screening was performed twice for the selection of a suitable mouse for fusion.
  • a Maxi Sorb plate was coated by adding 100 mI/well of 50 mM sodium carbonate coating buffer (pH 9.5) containing the free NLRP3 peptide at 1 pg/ml.
  • a separate coating solution was prepared by diluting APO-A1 in the same coating buffer at 1 pg/ml. These solutions were loaded onto the plate in alternating rows so as to provide two wells to load each sample that demonstrates a positive and negative result. This plate was incubated overnight at 4°C in static conditions.
  • RP was selected for fusion by expressing the most positive immune response.
  • a boost injection was given to BalbC mouse RP by injecting 100 mI of aliquoted IL-1 R at 200 pg/ml without any adjuvant.
  • SP2 cells were broken out from liquid nitrogen and were passaged in 10% FCS DMEM supplemented with 1 % Pen/Strep, 1 % L-glutamine until 3x12 ml T75 flasks were 75%- 90% confluent on the day of fusion.
  • FCS DMEM 10% FCS DMEM supplemented with 1 % Pen/Strep, 1 % L-glutamine until 3x12 ml T75 flasks were 75%- 90% confluent on the day of fusion.
  • SP2 cells were dislodged by tapping the flask and were centrifuged at 1000 rpm for 5 min at 37°C. The cells were resuspended in 20 ml SFM DMEM, centrifuged again, and resuspended in 10 ml SFM DMEM.
  • SP2 cells were stored in a Sterilin tube in SFM at 37°C, 6% C0 2 until needed.
  • the spleen was aseptically removed from the mouse that showed the strongest immune response.
  • Splenocytes were extracted by puncturing both ends of the spleen with a fine gauge needle and flushing 10-15 ml SFM DMEM.
  • Splenocytes were transferred to a sterilin tube and washed twice with 20 ml serum free DMEM by centrifugation at 1300 rpm for 5 min at 37°C and gently removing the supernatant.
  • the splenocytes were resuspended in 10 ml Serum free DMEM in a sterilin tube.
  • the SP2 cells were added to the splenocytes. This
  • SP2/splenocytes culture was centrifuged at 1300 rpm for 5 min at 37°C. After discarding the supernatant, 1 ml PEG was added to the SP2/splenocytes culture dropwise while stirring continuously over a period of 3 min. 1 ml SFM DMEM was added to the fusion mixture and stirred for 4 min. 10 ml SFM DMEM was added dropwise to the fresh culture and incubated for in 37°C water bath for 5 min. The cells were then centrifuged at 1000 rpm for 5 min at 37°C. The pellet was resuspended in 200 mL HATR media and was plated at 200 mI/well in 10x 96 well culture plates which were incubated 1 1 days at 37°C in 6% C0 2 prior to screening.
  • the samples from each dilution plate were loaded onto the ELISA plates at 100 mI/well by adding 1 row from each dilution plate per 2 rows on the ELISA plates to account for specificity of the coating antigens.
  • Two wells per ELISA were incubated with 100 mI 1xPBS as a negative control. These samples were incubated at 150 rpm for 2 hours at room temperature.
  • Both limiting dilutions were performed for 1-3 protoclones each by seeding 2-4x 96-well plates at 1 cell/ well in 200 mI culture/well. The plates were prepared by counting each culture in the 24-well plate and were diluted 10x as an intermediate dilution, then were diluted to 200 cells in 40 ml. The culture was plated at 200 mI/well and left to incubate at 37°C, 6% C0 2 for 7-10 days until the wells were 80%-90% confluent. Each well for both limiting dilutions were screened by ELISA as described in section 0.
  • each clone was left to grow in 37°C, 6% C0 2 for 6 days until each well became 80%-90% confluent.
  • each clone at 1 ml/well was transferred to a T25 flask containing 5 ml fresh 10% HATR DMEM for cryopreservation.
  • each 5 ml culture was centrifuged at 1000 rpm for 5 min at 37°C and was resuspended in 1 ml of fresh 10% DMEM HATR media.
  • Each 1 ml culture was transferred to a cryovial containing 300 mI of a 1 :1 ratio of FCS to DMSO. The vials were sealed and placed in a Mr. Frosty and transferred to the -70°C freezer for short-term storage.
  • Anti-NLRP3 produced from clone F226 7A7-1 E1-2D5 was selected for sequencing. Once the culture was confluent in the T25 flask, the supernatant was discarded. The cells were dislodged by cell scraping into 2 ml fresh media and were centrifuged at 7,600 rpm for 5 min at RT. The supernatant was then discarded and the pellet was flash frozen in liquid nitrogen and placed in -70°C until ready for mRNA extraction.
  • mice were immunised with NLRP3 peptide-KLH conjugate (designed by bioinformatics and synthesised by bioSynthesis Inc, Texas) and regular test bleeds were taken over an 1 1 week period. Test bleeds were then screened against the antigen.
  • mice Upon identification of positive mice, a fusion was performed and supernatant from hybridoma clones were then validated. The specific antibodies then underwent limiting dilution and cloning to produce a stable hybridoma cell line against NLRP3.
  • the antibodies were screened using ELISA against the target protein - NLRP3 - and clones with a signal of at least 3 times the background were selected. Antibodies from 24 clones were selected and further in house testing was performed to pick the best 6 clones.
  • 2RP was selected for the fusion of its splenocytes to fusion partner SP2 culture as it demonstrated the best immune response - see Figure 20.
  • Clones were selected from the post-fusion screening and were arrayed into a 24 well plate for expansion followed by a secondary screening that determines suitable protoclones for the first round of limiting dilutions. 3 clones were selected and limiting dilutions prepared - see Figure 22.
  • each clone was screened by ELISA against NLRP3 peptide and APO-A1.
  • the plates were screened by ELISA against NLRP3 peptide and APO-A1.
  • the 24 hybridoma populations producing the highest response and highest specificity were selected for expansion in a 24-well plate and cryopreservation - see Figure 25.
  • Western Blot Analysis is shown in Figure 27.
  • Western blots were performed using protein lysates from THP-1 macrophages to test supernatant containing anti-NLRP3 monoclonal antibody collected from the best 6 clones from a fusion hybridoma cell line untreated (lane 1 ) and stimulated with LPS and ATP (lane 2, (protein ladder lane 3)).
  • Clone 18 was selected for sequencing and was used in the bispecific monoclonal antibody development.
  • the aim was to produce a range of antibodies against NLRP3 that were functional in preventing assembly of the NLRP3 inflammasome.
  • 2RP was selected for fusion.
  • 24 monoclonal hybridoma cell lines were produced from two rounds of limiting dilutions. Each population was selected by highest production and highest specificity for NLRP3.
  • the clone F226 7A7-1 E1-2D5 was shown to be most active in preventing NLRP3 assembly in the in vitro assay. These final cell lines have been frozen down, and the antibody expressed by this 7A7- 1 E1-2D5 will be sequenced for the next stage in the production of the bi-specific, InflaMab.
  • Example 6 NLRP3 monoclonal sequencing mRNA was extracted from the hybridoma cell pellets on 23/02/16. Total RNA was extracted from the pellets using Fusion Antibodies Ltd in-house RNA extraction protocol (see Example 3).
  • cDNA was created from the RNA by reverse-transcription with an oligo(dT) primer. PCR reactions are set up using variable domain primers to amplify both the VH and VL regions of the monoclonal antibody DNA giving the following bands (see Figure 28):
  • the VH and VL products were cloned into the Invitrogen sequencing vector pCR2.1 and transformed into TOP10 cells and screened by PCR for positive transformants. Selected colonies were picked and analyzed by DNA sequencing on an ABI3130xl Genetic Analyzer, the result may be seen below.
  • VH1.1 (SEQ ID NO: 33)
  • VH3.4 (SEQ ID NO: 35)
  • VH3.1 (SEQ ID NO: 36)
  • VH3.5 (SEQ ID NO: 36)
  • VH3.8 (SEQ ID NO: 36)
  • variable domain is highlighted in BOLD.
  • CDRs Complementarity Determining Regions
  • Blue shaded circles are hydrophobic (non-polar) residues in frameworks 1-3 at sites that are hydrophobic in the majority of antibodies.
  • Red amino acids in the framework are structurally conserved amino acids.
  • VL1.1 (SEQ ID NO: 37)
  • VL1.6 (SEQ ID NO: 38)
  • VL1.2 (SEQ ID NO: 39)
  • VL1.7 (SEQ ID NO: 40)
  • VL1.4 (SEQ ID NO: 41)
  • VL1.5 (SEQ ID NO: 42)
  • SPSVTLFPPSTEELSL (SEQ ID NO: 43) The variable domain is highlighted in BOLD.
  • CDRs Complementarity Determining Regions
  • Blue shaded circles are hydrophobic (non-polar) residues in frameworks 1-3 at sites that are hydrophobic in the majority of antibodies.
  • Red amino acids in the framework are structurally conserved amino acids.
  • variable domain sequences of the monoclonal antibodies IL-1 R1 and NLRP3 were sequenced.
  • the antibody was constructed using the IL-1 R1 antibody with an lgG2a mouse constant domain sequence.
  • a short linker was added to the C-terminal end of the heavy chain and the NLPR3 variable domains in an ScFv format with the linker (GGGGS) 3 was attached to create the bispecific.
  • the DNA and amino acid sequences can be found below.
  • the constructs were cloned into ATUM vector pD2610-v5 and verified by sequencing.
  • Figure 31 illustrates the bispecific design and the plasmid map of InflaMab.
  • CAAG ACT G AGG ACACTGCCACCT ACTT CT GTGCCAAG AG CGTGT ACTT CAACTGG AG AT ACTT C
  • Example 8 InflaMab (Bispecific against IL-1R1 and NLRP3) transient expression
  • InflaMab is a 210 kiloDalton (kDa) bispecific mouse antibody composed of two pairs of light chain and two pairs of heavy chains with ScFv domains fused to the N-terminal, complexed together via disulphide bonds.
  • a mammalian expression vector encoding InflaMab was transfected into ExpiCHO cells.
  • the expressed antibody was subsequently purified from clarified culture supernatant via protein A affinity chromatography. The concentration of purified antibody was measured using a NanoDrop Lite, Thermofisher and purity was evaluated using SDS-PAGE.
  • Suspension adapted ExpiCHO cells were routinely cultured at 1.0-3.0 x10 5 cells/ml every 2-3 days in 500ml vented Erlenmeyer flasks (Corning, Netherlands) agitated at 135 rpm at 37°C 8% C0 2 .
  • Plasmid DNA for transfection was isolated using a Purelink Hipure plasmid filter maxiprep kit (Thermo Fisher, UK) as per the manufacturer instructions. DNA was quantified using a Nano Drop life spectrophometer as per the manufacturer instructions.
  • ExpiCHO cells Twenty-four hours prior to transfection, ExpiCHO cells were seeded at a concentration of 4.0 x 10 6 cells/ml in ExpiCHO expression medium and grown overnight at 135 rpm, 37°C 8% C0 2 . On the day of transfection, 250ml ExpiCHO cells were diluted to a final density of 6.0 X 10 6 cells/ml in ExpiCHO expression medium. 1 .Opg/rnl of plasmid DNA and 0.32% (v/v) Expifectamine CHO reagent (Thermo Fisher) were diluted separately in 4% (v/v) OptiPro SFM (Thermo Fisher).
  • the Expifectamine CHO/Optipro complex was added to the Plasmid DNA/Optipro complex dropwise. The transfection mixture was immediately added to the ExpiCHO cells. Transfected cells were incubated overnight at 135rpm, 37°C, 8% C0 2 .
  • AKTA GE Healthcare
  • Purifications were performed using AKTA (GE Healthcare) chromatography equipment. Prior to use, all AKTA equipment was thoroughly sanitized using 1 M NaOH. Following centrifugation, filtered (0.22 pm) cell culture supernatant was applied to an AKTA system fitted with a 1 ml HiTrap Protein A column (equilibrated with wash buffer). Following loading, the column was washed with 20 column volumes of wash buffer. Bound antibody was step eluted with 10 column volumes of elution buffer. All eluted fractions were neutralised with Tris pH 9.0 buffer. Eluted fractions corresponding to elution peak were selected for overnight dialysis into PBS. The purity of the antibody was >95%, as judged by SDS-polyacrylamide midi gels.
  • SDS PAGE Sodium Dodecyl Sulphate Polyacrylamide Electrophoresis
  • InflaMab is >95% pure as judged by analysis of SDS-polyacrylamide gels. Under reducing conditions, both heavy and the light chains of the antibody are visible and are observed at the expected molecular weight of approximately 80 and 27kDa, respectively. Under non-reducing conditions, a single major band and several minor bands are observed. The additional bands (impurities) are likely the result of non-glycosylated IgG and IgG degradation products (e.g. a single [partial] light chain, a combination of two heavy and one light chain, two heavy chains, two heavy and one light chain).
  • THP1 cells for Figure 33a and b, were cultured in 96-well plates at 100,000 cells/200pl complete media. PMA (100pg/ml for 72 hours) was used to differentiate THP-1 cells into macrophages.
  • THP1 cells were stimulated with LPS (1 pg/ml) for 3 hours, treated with MCC950 (1 mM) or the IL- 1 R1/NLRP3 Ab in a dose dependent fashion from 0.0025ng/ml to 2.5ng/ml for figure 33a or with IL- 1 R1/NLRP3 Ab (1 nM) for figure 33b or with IgG control antibody for 30 minutes, followed by ATP (5mM) for 1 hour.
  • IL-1 b release was measured in the supernatant by ELISA. Inflamab prevents caspase-1 activation in THP1 cells - see Figure 34.
  • THP1 cells, for Figure 34 were cultured in 96-well plates at 100,000cells/200ul complete media.
  • PMA 100ug/ml for 72 hours was used to differentiated THP-1 cells into macrophages. Following 24 hours of rest, differentiated THP1 cells were stimulated with LPS (1 ug/ml) for 3 hours, treated with the IL- 1 R1/NLRP3 Ab (1 ug/ml) for 30 minutes, followed by ATP (5mM) for 1 hour. Caspase-1 activation was assessed by staining cells with a non-cytotoxic Fluorescent Labelled Inhibitor of Caspase-1 (FAM-FLICA) and DAPI (nuclear stain).
  • FAM-FLICA non-cytotoxic Fluorescent Labelled Inhibitor of Caspase-1
  • DAPI nuclear stain
  • THP1 cells for Figure 35, were cultured in 96-well plates at 100,000cells/200ul complete media. Differentiation of THP1 cells was induced by PMA (100ug/ml for 72 hours). Following 24 hours of rest, differentiated THP1 cells were stimulated with LPS (1 ug/ml) for 3 hours, treated with a pHrodo red labelled IL-1 r/NLRP3 Ab (1 ug/ml) for 30 minutes, followed by ATP (5mM) for 1 hour. The internalization of the Ab was tracked using a pHrodo red labelled bi-specific Ab that only fluoresces when internalized.
  • a representative confocal image shows the internalization of the pHrodo red labelled bi-specific Ab in a differentiated THP1 cell.
  • B A representative confocal image shows significant reduction of caspase-1 activation (green) in THP1 cells that have internalized the bi-specific Ab (red, white arrow) as compared to THP1 cells that did not internalize the Ab (green only).
  • Glaucoma is the leading cause of irreversible blindness worldwide, characterized by the progressive loss of retinal ganglion cells (RGCs).
  • RRCs retinal ganglion cells
  • IOP intraocular pressure
  • IOR-independent mechanisms also contribute to the development and progression of glaucoma and highlight the need for developing IOR-independent neuroprotective therapies to prevent disease progression and preserve vision.
  • Glaucoma is a complex multifactorial disease and while the exact mechanisms that mediate axon degeneration and death of RGCs are not well understood, there is growing evidence that axon damage in the optic nerve head (ONH) precedes death of the RGCs [7, 8]. Moreover, the axon damage in the ONH has been linked to glial activation and inflammation [9, 10].
  • activated astrocytes [10, 1 1] and activated microglia [9, 12] are detected in ONH and coincides with increased expression of proinflammatory cytokines such as IL- 1 b and TNFa and neurotoxic mediators such as Nitric Oxide (NO), Reactive Oxygen Species (ROS), and Glutamate [12-14].
  • proinflammatory cytokines such as IL- 1 b and TNFa
  • neurotoxic mediators such as Nitric Oxide (NO), Reactive Oxygen Species (ROS), and Glutamate [12-14].
  • NO Nitric Oxide
  • ROS Reactive Oxygen Species
  • Glutamate Glutamate
  • the NLRP3 inflammasome is an intracellular multi-protein complex that triggers inflammation in response to signals generated by infectious organisms, as well as endogenous signals associated with cell stress and tissue damage [15]. Dysregulation of the NLRP3 inflammasome has been implicated in several neurodegenerative diseases, including Alzheimer’s disease and multiple sclerosis [16] but, most recently, activation of the NLRP3 inflammasome has been associated with the death of RGCs following retinal ischemia reperfusion injury and optic nerve crush [17, 18].
  • MCC950 pharmacologically targeting NLRP3 can serve as a neuroprotective therapy in glaucoma.
  • MCC950 due to a very short half-life, MCC950 had to be administered systemically every other day for the length of the study and as a potential treatment for glaucoma, systemically blocking the NLRP3 inflammasome in an aging population is not ideal, since the inflammasome plays an integral role in host defense against infection [15, 22].
  • RGC function is currently being measured by pERG using a fully integrated ERG system for rodents (Celeris). Changes in pERG amplitude is being measured in mice that received microbeads only and compared to mice that received microbeads plus Inflamab (final vitreous concentration of 2.5, 25, and 250 ng/ml) or saline only (no beads) [ Figure 42]. In these studies, preliminary pERG results reveal a recovery of RGC function in the microbead-injected mice treated with Inflamab at a final vitreous concentration of 250 ng/ml as compared to mice receiving microbeads only or microbeads plus Inflamab at 2.5 or 25 ng/ml.
  • Visual acuity using an optomotor reflex-based spatial frequency threshold test, as well as RGC and axon quantification, is performed to further corroborate the neuroprotective effect of Inflamab and including additional control groups (microbead-injected mice treated with the appropriate IgG control antibody).
  • NLRP3 is constitutively expressed in the mouse and human optic nerve head - see Figure 36.
  • NLRP3 is constitutively expressed in optic nerve head astrocytes of normal (nonglaucomatous) human eyes - see Figure 37.
  • Immunofluorescence in sections of human optic nerve shows co-localization of NLRP3 (red) with the astrocyte-specific marker GFAP (Green) in the unmeylinated lamina cribosa region of the optic nerve head.
  • Dapi blue was used to identify all nucleated cells. Images are representative of staining performed on optic nerve sections obtained from three individual (non-glaucomatous) human optic nerves.
  • NLRP 3 inflammasome assembly in the ONH coincides with induction of inflammatory mediators at 7 days post microbead injection - see Figure 38.
  • ASC-speck formation was monitored in vivo using a fluorescent reporter mouse (ASC citrine/Cre+).
  • ASC citrine/Cre+ ASC citrine/Cre+.
  • ASC citrine/Cre+ ASC citrine/Cre+.
  • ASC citrine/Cre+ ASC citrine/Cre+.
  • A At 7 days post microbead injection or saline as a control, frozen eye sections were stained for GFAP (astrocytes, pink), MBP (myelin, red), and DAPI (blue).
  • A Frozen sections (3 sections per eye) were taken from WT and ASC KO eyes at 0, 7, and 14 days after microbead injection and the total number of Iba1 + cells (macrophage/microglia) was counted in the ONH region (top of the ONH to the myelinisation zone).
  • B Results show a significant increase in the number of Iba1 + cells at D7 and D14 post microbead injection in WT but not ASC KO ONH.
  • InflaMab NLRP3/IL1 R1 bispecific antibody
  • WT C57BL/6J mice received a single intravitreal injection of Inflamab (final vitreous concentration of 2.5, 25, or 250 ng/ml) on Day 0, immediately preceding the injection of microbeads. Mice receiving saline only (no beads) served as a normal (no glaucoma) control.
  • Visual acuity using an optomotor reflex-based spatial frequency threshold test, as well as RGC and axon quantification will also be performed to further corroborate the neuroprotective effect of Inflamab and additional control groups (microbead-injected mice treated with IgG control) will also be added.

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